Fish cytokines: current research and applications

Cytokines are low molecular weight glycoproteins involved in the regulation of the immune system, and more than 100 cytokines have hitherto been identified in humans. Cytokines similar to those found in humans have also been found in fish. The innate immune response of fish can be examined by using cytokines as markers. We developed a multiplex reverse transcription–polymerase chain reaction assay to analyze the expression of various cytokine genes. Using this assay, we were able to investigate the fish immune response when it had been activated by immunostimulants. The involvement of inflammatory cytokines, such as interleukin-1β, in the immune system of fish was revealed following administration of an immunostimulant.


Introduction
A major concern in aquaculture is the occurrence of fish diseases, which have hitherto mainly been controlled by administering antibacterial agents. However, because of the emergence of drug-resistant bacteria and problems associated with food hygiene as a consequence, it is preferable to avoid treating fish diseases with antibacterial drugs (Sakai 1999).
The immune system of higher vertebrates comprises innate and acquired immunity. Acquired immunity involves the recognition of foreign antigens via antigen receptors on T-and B-lymphocyte membranes. These antigen receptors are activated via antigen stimulation and have high affinity to a specific antigen. The establishment of acquired immunity usually requires several days, and a rapid immune response to, for example, microbial infection is not always initiated (Nakanishi et al. 2018). On the other hand, an innate immune response is required at the early stages of infection and is mainly attained through the action of phagocytes and humoral factors (Nakanishi et al. 2018).
Innate immunity is conventionally regarded as more important than acquired immunity in preventing infectious diseases in fish. Cytokines are mainly secreted by cells of both the innate and acquired immune systems. These molecules effect small physiological changes via specific receptors present on target cell surfaces and are responsible for signal transmission between cells (Zou and Secombes 2016). Cytokines play an important role in the innate immune response of fish. In this review, recent research on cytokine genes is presented, with a specific focus on describing the molecules important to the innate immune responses of fish, and the immunostimulants that elicit them.

Interleukin-1 family
Interleukin (IL) 1 is a pro-inflammatory cytokine that plays an important role in innate immunity. In addition to IL-1, around 100 types of IL are presently known in humans. Recent genomic studies have identified IL-1 as the evolutionarily earliest cytokine in fish (Zou et al. 1999;Wang et al. 2009). In mammals, the genes coding for IL-1α and IL-1β are found on the same chromosome and are adjacent to one another, whereas in fish, only the gene encoding for IL-1β has so far been identified. Using DNA vaccination, Kono et al. (2002), reported that IL-1 plays an important role in fish immunity by activating lymphocytes and phagocytic cells and increasing resistance to Aeromonas hydrophila infection. Recently, a newer type of il1b gene was discovered in Japanese medaka Oryzias latipes. This gene is 76.0% homologous to the previously identified il1b gene, and is present on different chromosomes (Morimoto et al., personal communication). Furthermore, this gene has been confirmed to have pro-inflammatory actions.

IL-2 family
In the IL-2 family, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 are found in humans (Lin and Leonard 2018). IL-2, IL-4/13A, IL-4/13B, IL-7, IL-15, and IL-21 have been reported in fish (Zou and Secombes 2016). As in mammals, the genes of IL-2 and IL-21 in fish are found in tandem (Bird et al. 2005). Two IL-4/13 genes in fish, il4/13a and il4/13b, have been found to be counterparts of mammalian IL-4 and IL-13, and are thought to have arisen through genome duplication. Moreover, the presence of IL-15 was revealed for the first time in Japanese pufferfish Takifugu rubripes, and another il15 ortholog similar in structure to il15 has been confirmed in Japanese pufferfish and zebrafish Danio rerio (Gunimaladevi et al. 2007).

IL-6 family
The molecules identified in humans which belong to the IL-6 cytokine family are IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF). The IL-6 sub-family cytokines are major players in hematopoiesis, and have pro-and anti-inflammatory properties. IL-6, IL-11 and CNTF-like occur in fish, and M17 has been identified as a fish-specific molecule that could possibly be an ancestral molecule to LIF and OSM (Hanington and Belosevic 2007). Although the functions of these molecules have not been clarified, recombinant IL-6 has been reported to promote phagocyte proliferation in rainbow trout (Costa et al. 2011). The rIL-6-induced transient expression (up to 4 h post-stimulation) of SOCS1-3 and interferon (IFN) regulatory factor (IRF)-1, and more sustained upregulation of antimicrobial peptide (AMP) gene expression at a similar level, were found in primary (head kidney derived) macrophage cultures (Costa et al. 2011). , and IFN-γ are cytokines of the IL-10 family. In general, fish IL-10, similarly to mammalian orthologs, acts as a suppressor and exerts a conserved role in dampening inflammatory responses. The functions of IL-10 have been characterized in goldfish and carp (Piazzon et al. 2015). In fish, il10 and il19/20 genes are present in tandem on the same chromosome and code for IL-22, IL-24, and IFN-γ . We discovered that a newly identified IFN-γ gene (ifngrel) exists next to the ifng gene (Igawa et al. 2006). IFN-γ rel is thought to be involved in immune defense against bacterial infection. Elevated expression of the ifngrel gene in the carp, rohu Labeo rohita, has been reported following infection of the fish with A. hydrophila, Edwardsiella tarda, or Shigella flexneri (Swain et al. 2015).

Chemokine family
Chemokines are produced in large quantities in areas of inflammation and induce the migration of leukocytes from blood vessels to inflamed tissues. To date, more than 50 chemokines have been identified in humans. Chemokines have four highly conserved cysteine residues and are classified into four subfamilies, namely CXC, CC, CX3C, and XC, according to the motif formed by the two N-terminal cysteine residues. Many chemokines have also been identified in fish. More than 100 types of zebrafish chemokines have been reported, although none that belong to the CX3C subfamily (Nomiyama et al. 2008). Several CXC chemokines, including CXCL8_L1 (IL-8), CXCL11_L1 (γIFN-inducible protein), CXCL_F4 and CXCL_F5 were found to increase in rainbow trout cell lines (i.e., RTG-2 and RTS-11 cells) after rIL-1β stimulation (Chen et al. 2013).

Interferon family
Interferons are types of cytokines produced by animal cells in response to the invasion of viruses, heterologous RNA, and certain sugars. Cells infected with viruses are known to exhibit an interference phenomenon that blocks infection by other viruses. Human IFNs include type I IFNs (13 types of IFN-α, IFN-β, IFN-ω, IFN-ε, and IFN-κ), type II IFN (IFN-γ), and type III IFNs (IFN-λ1, IFN-λ2, and IFN-λ3). In fish, type I IFNs (homologs to the human IFN-α and IFN-β) exhibit antiviral activity, and type II IFN (IFN-γ) have also been reported to exhibit bactericidal activity against intracellular parasitic bacteria (Zou and Secombes 2016). A type I IFN in fish has been shown to have antiviral activity, but the gene encoding it has introns and exons, unlike mammalian genes coding for IFN (Zou and Secombes 2016).

TNF family
The TNF family includes proteins with the following motifs: . More than 20 TNF molecules have been reported in humans, including TNF-α, lymphotoxin (LT)-α cytokines involved in apoptosis, the Fas ligand, the TNF-related apoptosis-inducing ligand (TRAIL), the receptor activator of nuclear factor-κ B (RANK) ligand,the TNF receptor superfamily member 4 (OX40) ligand, and proliferation-inducing ligand 20 (TNF ligand superfamily member 13; APRIL 20). At least ten TNF genes have been identified in pufferfish (Biswas et al. 2015). TNF-α is the most immunologically important molecule in the TNF family. In humans, three genes, tnfa, tnfb (also called lta ), and ltb exist in tandem. In fish, two genes, tnfa and tnfn are duplicated in tandem on the chromosome (Savan et al. 2005). In addition, unlike mammals, fish have another tnfa on another chromosome (Kinoshita et al. 2014). It has been suggested that members of the fish TNF-α family are involved in the regulation of leukocyte homing, proliferation and migration. Constitutive expression of TNF-α was detected in the trout thymus, which suggests that it could play a role in promoting thymocyte growth (Hino et al. 2006). Members of the TNF-α family in fish exert pro-apoptotic activity, as do their mammalian homologues. High doses (> 400 ng/mL) of Chinese perch TNF-α induced apoptosis in human Hela cells (Xiao et al. 2007). In tilapia, TNF-α has been shown to upregulate granzyme expression in non-specific cytotoxic cells and to protect these cells from activation-induced cell death (Praveen et al. 2006).

Other cytokines
Other important cytokines in fish include the IL-12 subfamily molecules, IL-18, transforming growth factor (TGF)-β, and macrophage colony-stimulating factor (M-CSF) (Zou and Secombes 2016). The function of fish IL-12 has been studied in two different paralogues of the p40 chain heterodimerized with a common p35. In grouper, rIL-12 increased the concanavalin A (ConA)-induced proliferation of peripheral blood lymphocytes and tnfa expression (Tsai et al. 2014). In amberjack Seriola dumerili, rIL-12-mixed Nocardia seriolae-FKC vaccine induced cell-mediated immunity and production of type 1 helper (Th1) cells with antigen memory against N. seriolae infection (Matsumoto et al. 2017). IL-18, as well as IL-1β, is released after cleavage by caspase-1 following stimulation of inflammasome with pathogen-associated molecular patterns or damageassociated molecular patterns. Unlike IL-1β, which is transiently induced by inflammatory stimuli, IL-18 is constitutively expressed in tissues of the innate and adaptive immune defense systems in fish (Zou et al. 2004). Most studies on TGF-β in fish have focused on its function, in combination with other cytokines, as a driver of Th17 differentiation (e.g., IL-6), but it is also a key immunosuppressive cytokine secreted by regulatory T cells (Haddad et al. 2008;Wei et al. 2015). Fish M-CSF was first identified from goldfish (Barreda et al. 2005), and was shown to induce the differentiation of monocytes into macrophages and the proliferation of monocyte-like cells with a mononuclear phagocyte function (Grayfer et al. 2009).

Immune function enhanced by immunostimulants
Many immunostimulants have been developed for use in aquaculture, the major ones of which are shown in Table 1 (Sakai 1999). Immunostimulants are generally considered to prevent disease by increasing the innate immune response; we describe here the immune mechanisms that they activate in fish.

Activation of phagocytes
Phagocytic cells are functionally activated by the administration of immunostimulants. The functions of phagocytic cells can be classified as migratory, phagocytic, and bactericidal, with phagocytosis the one most intensively investigated in immunostimulation research. Generally, the phagocytosis rate and phagocytic index of the renal and intraperitoneal phagocytes of fish treated with immunostimulants, e.g., levamisole (Kajita et al. 1990), and lactoferrin , significantly increases. Furthermore, the phagocytosis rate and phagocytic index increased significantly after phagocytes had been cultured in vitro in a medium containing an immunostimulant (Sakai et al. 1995a, b).
The sterilizing pathways of phagocytic cells can be broadly divided into two types: systems that depend mainly on oxygen and those that do not. Oxygen-dependent systems basically comprise oxygen compounds (e.g., O 2 and H 2 O 2 ) and nitrogen compounds (e.g., NO, NO 2 , N 2 O 3 , and NO 2 + ). Bactericidal substances, such as lysozyme and defensin, are important oxygen-independent systems. The administration of chitin, lactoferrin, or levamisole as immunostimulants enhanced the bactericidal action of oxygen compounds and oxygenated compounds (superoxides) produced by phagocytes (Kajita et al. 1990;Sakai 1999;Sakai et al. 1992). The migration ability of phagocytic cells was reported to significantly increase in channel catfish fed with yeast glucansupplemented feed (Duncan and Klesius 1996).

Activation of lymphocytes
Lymphocytes have also been reported to be activated by the administration of immunostimulants. A lymphocyte mitogenic response was promoted in rainbow trout (Oncorhynchus mykiss) leukocytes cultured in a culture medium [with ConA and lipopolysaccharide (LPS)] containing firefly squid (Watasenia scintillans) extract in vitro; the production of macrophage-activating factor increased significantly (Siwicki et al. 1996). Similar results have been obtained with a growth hormone and glycyrrhizin (Jang et al. 1995;Sakai et al. 1996).

Enhancement of complement activation
The complement system is a series of enzyme systems that can destroy foreign substances either alone or in coordination with antibodies. The activation of complement systems can enhance the phagocytic ability of phagocytes, and immunostimulants can increase this activation. Engstad et al. (1992) reported an increase in a complement factor  DNA Tassakka and Sakai 2004 in Atlantic salmon (Salmo salar) injected with yeast glucan. Similar results have been reported in channel catfish (Ictalurus punctatus) (Li and Lovell 1985) and Atlantic salmon (Hardie et al. 1991) treated with high doses of vitamin C, and rainbow trout (Kajita et al. 1990) treated with levamisole.

Activation of lysozyme
Lysozyme is a heat-resistant antibacterial enzyme found in the serum and body surface mucus of fish. Glucans are generally known to increase lysozyme activity (Engstad et al. 1992). However, lysozyme activity did not increase in fish in response to the administration of chitin (Sakai et al. 1992) or lactoferrin (Sakai et al. 1995a, b). Fish have two types of lysozyme genes: chicken-type (C-type) and goose-type (G-type) (Callewaert and Michiels 2010;Hikima et al. 2002). Lytic activity of fish lysozyme has been detected in skin mucus, serum, the kidneys (including head and trunk kidneys), liver, gills, and eggs (Saurabh and Sahoo 2008;Yano 1995). The expression of the lysozyme gene in these tissues was shown by Callewaert and Michiels (2010) and Hikima et al. (2002). The messenger RNA levels of C-type and G-type lysozymes are upregulated in many fish species following challenge with immunostimulants and pathogenic microbes (Hikima et al. 1997;Jiménez-Cantizano et al. 2008;Ye et al. 2010). In turbot Scophthalmus maximus, two G-type lysozyme genes have been identified, one of which showed significant upregulation in the intestine following infection with Vibrio anguillarum and Streptococcus iniae (Gao et al. 2016). Furthermore, recombinant fish C-type lysozymes possess bacteriolytic and/or bactericidal activities against several Gram-positive and Gram-negative bacterial pathogens Hikima et al. 2001;Ye et al. 2010;Gao et al. 2012).

Disease prevention with immunostimulants
Diseases that can be prevented by the use of immunostimulants include streptococcosis, vibriosis, and furunculosis, all of which are caused by extracellular bacteria. Few cases of immunostimulants being effective against viral diseases or intracellular bacteria, such as bacteria responsible for bacterial kidney disease, have been documented. In most cases, activated phagocytes are effective against extracellular bacteria. The activation of IL-1β and TNF-α is indispensable for this because their activation effects several innate immune responses including phagocytosis (Sakai 1999). The activation of type-I IFN is most effective against viral infections. In Japanese flounder Paralichthys olivaceus, rIFN-γ is effective in reinforcing immune responses and preventing edwardsiellosis (Jung et al. 2012).

Multiplex reverse transcription-polymerase chain reaction assay
The multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay is designed to simultaneously amplify multiple genes during one test in one reaction tube (Bonetta 2006). We performed multiplex RT-PCR assay using chimeric primers for each gene designed by GenomeLab GeXP (Beckman Coulter), and quantified each gene. As the level of gene expression is investigated by amplifying and analyzing multiple genes in one tube with this method, both the duration and cost of analysis could be greatly reduced, enabling comprehensive examination of the expression levels of various genes.
In this multiplex RT-PCR reaction, each chimeric primer (i.e., a primer composed of both a gene-specific and universal sequences) was constructed so that the PCR products had different sizes. The PCR products of each gene were synthesized by using these chimeric primers so that universal sequences were added to both the 5′ and 3′ ends during the reverse transcription reaction and several PCR cycles. Thereafter, a universal primer labeled with a fluorescent dye was used during synthesis, so that a fluorescent PCR product was produced. The PCR products were fractionated based on size via capillary gel electrophoresis, and the gene expression level was calculated by detecting the fluorescence intensity of each gene.

Development of a multiplex RT-PCR assay to quantify fish cytokine genes
We successfully developed a multiplex RT-PCR assay for the comprehensive quantification of fish cytokine gene expression. We clarified the existence of, and cloned, various cytokine genes from the Japanese pufferfish T. rubripes (Table 2) (Kono et al. 2013).

Expression of cytokine genes in immunostimulant-treated or bacteria-infected fish
The effectiveness of fish immunostimulants was examined using the above-mentioned cytokine-multiplex RT-PCR assay. Table 3 shows the changes over time in the expression of various cytokine genes, with LPS causing a strong inflammatory reaction and polyinosinic-polycytidylic acid [poly(I:C)] exhibiting antiviral activity. In pufferfish treated with LPS, the expression of IL-1β, TNF-α, IL-6, and IL-17A genes as pro-inflammatory cytokines was confirmed to increase 4-8 h after administration. Increased expression of IL-4/13A and IL-12p35 genes was also observed. At 24 h after LPS administration, the expression of the IL-10 gene increased, whereas that of IL-1β and TNF-α genes decreased to pre-stimulation levels. An increase in the expression of type-I IFN genes was not confirmed. In cells treated with poly(I:C), an increased expression of IL-6, TNF-α, and other genes was observed, similar to that for LPS administration, but with a marked increase in type-I IFN gene expression. Thus, LPS and poly(I:C) activated different cytokines.
The cytokine responses in pufferfish infected with E. tarda are shown in Fig. 1. Increased IL-21 expression was observed on the first day after infection, and TNF-α expression levels were increased on the fifth day. In contrast, the expression of IL-1β, IL-10, IL-17A, type-I IFN, and IL-4/13A genes decreased 7 days after infection.

Conclusion
In this review, we have described the roles of cytokines and their use as indices of innate immunity in fish, and introduced methods that can be used to detect them (e.g., the multiplex RT-PCR assay). However, research to elucidate the functions of cytokines in fish has only recently begun, and further work is necessary for the selection of, for example, appropriate immunostimulants and vaccine adjuvants for the prevention of infections in farmed fish.
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